羊消化道4种易混淆线虫虫卵的形态学观察与比较

Morphological observation and comparison of the eggs of four confusable nematodes from the digestive tract of sheep

为了方便临床上鉴定羊消化道常见且易混淆线虫虫卵并丰富相关资料,试验从羊消化道中采集疑似捻转血矛线虫、夏伯特线虫、食道口线虫和奥斯特线虫共4种线虫,采用形态学和分子生物学方法鉴定虫种,将4种线虫于体外短暂培养后得到纯种虫卵,通过观察虫卵不同发育阶段的胚胎形态变化获取每种线虫虫卵形态特征,并对虫卵的长径、宽径和长宽比进行测定。结果表明:经鉴定4种线虫分别为捻转血矛线虫、绵羊夏伯特线虫、粗纹食道口线虫和普通奥斯特线虫。4种线虫虫卵初期胚胎多呈收缩状态且形似桑葚,在外观形态上具有相似性,均为双层卵壳,多数对称,少数不对称。捻转血矛线虫虫卵呈短椭圆形,颜色较浅;绵羊夏伯特线虫虫卵呈椭圆形,颜色较捻转血矛线虫虫卵深;粗纹食道口线虫虫卵呈长椭圆形,颜色较捻转血矛线虫虫卵深,与绵羊夏伯特线虫虫卵相近;普通奥斯特线虫虫卵亦呈长椭圆形,颜色与捻转血矛线虫相近,较绵羊夏伯特线虫和粗纹食道口线虫虫卵浅。虫卵在生理盐水中培养1~6 d后,捻转血矛线虫、绵羊夏伯特线虫和普通奥斯特线虫依次进入蝌蚪期和幼虫期,但仅凭肉眼依然难以区分虫卵种类;绵羊食道口线虫未观察到含蝌蚪或含幼虫的虫卵,仅在桑葚期观察到胚胎收缩成1团及分裂成2团或3团不同形态的虫卵。部分粗纹食道口线虫和普通奥斯特线虫虫卵可观察到明显的气室。捻转血矛线虫卵大小为(61.22~102.05)μm×(37.96~69.33)μm,长宽比为1.51;绵羊夏伯特线虫卵大小为(81.89~139.14)μm×(43.97~90.60)μm,长宽比为1.57;粗纹食道口线虫卵的大小为(84.57~104.59)μm×(40.86~59.18)μm,长宽比为1.83;普通奥斯特线虫卵大小为(82.83~117.22)μm×(41.70~71.79)μm,长宽比为1.76。说明4种线虫虫卵的外观形态虽有细微差异,但没有特征性标识,临床上易造成误判,因此采用分子生物学方法进行鉴定更为可靠。

In order to facilitate the clinical identification of common and confusing nematode eggs in the digestive tract of sheep and to enrich related information,in the experiment,four species of nematodes,suspected to be Haemonchus contortus,Chabertia ovina,Oesophagostomum,Ostertagia circumcincta,were collected from the digestive tract of sheep.Morphological and molecular biological methods were used to identify the nematode species;pure eggs of the four nematodes were obtained after short-term culture in vitro;morphological characteristics of each nematode egg were obtained by observing the morphological changes of the embryos at different developmental stages of the eggs,and the length diameter,width diameter and ratio of length to width of eggs were measured.The results showed that four nematodes were identified as the eggs of Haemonchus contortus,Chabertia ovina,Oesophagostomum asperum and Ostertagia circumcincta,respectively.The initial embryos of the four species of nematode eggs were mostly contracted and mulberry-like,with similarities in appearance and morphology,all of which had double egg shells,with most being symmetrical and a few being asymmetrical.The eggs of Haemonchus contortus were short oval and light in color.The eggs of Chabertia ovina were ellipsoid and darker than those of Haemonchus contortus.The eggs of Oesophagostomum asperum were oblong and darker than the eggs of Haemonchus contortus,which were similar to the eggs of Chabertia ovina.The eggs of Chabertia ovina were also oblong,similar in color to that of Haemonchus contortus,and lighter than the eggs of Chabertia ovina and Oesophagostomum asperum.After incubating the eggs in saline for 1-6 d,Haemonchus contortus,Chabertia ovina and Ostertagia circumcincta entered the tadpole and larval stages sequentially,but it was still difficult to differentiate between the egg types by the naked eye alone.No eggs containing tadpoles or larvae were observed in Oesophagostomum asperum and only in the mulberry stage were eggs with different morphologies of embryos contracting into one mass and splitting into two or three masses observed;obvious air chambers were observed in some eggs of Oesophagostomum asperum and Ostertagia circumcincta.The egg size of Haemonchus contortus was(61.22-102.05) μm×(37.96-69.33) μm,and the aspect ratio was 1.51.The egg size of Chabertia ovina was(81.89-139.14) μm×(43.97-90.60) μm,and the aspect ratio was 1.57.The egg size of Oesophagostomum asperum was(84.57-104.59) μm×(40.86-59.18) μm,and the aspect ratio was 1.83.The egg size of Ostertagia circumcincta was(82.83-117.22) μm×(41.70-71.79) μm,and the aspect ratio was 1.76.The results indicated that the eggs of the four nematode species had no characteristic markers,although there were subtle differences in their appearance and morphology,which was easy to cause misjudgment in clinic and were therefore more reliably identified by molecular biology methods.